Proteome analysis elucidating post-mortem changes in cod (Gadus morhua) muscle proteins

Proteome analysis was successfully applied to study the alterations in fish muscle proteins during ice storage. The processes occurring during post-mortem metabolism are known to lead to characteristic changes in the texture and taste of fish muscle. Endogenous proteases are anticipated to play the major role in these processes, although the exact mechanisms during fish meat tenderization have yet to be depicted. Protein changes in cod (Gadus morhua) muscle were followed during 8 days of storage. Within the partial proteome (pI 3.5-8.0, MW 13-35 kDa) significant changes were found in 11 protein spots. In nine protein spots the intensity increased, and for eight of these the increases were significant (p < 0.05) within the first 2 h post-mortem. In contrast, two protein spots decreasing in intensity showed significant (p < 0.03) changes after 8 days, thereby indicating that in the fish muscle different biochemical processes are involved in the protein changes observed post-mortem.     

Incorporation and stabilization of omega-3 fatty acids in surimi made from cod, Gadus morhua

Surimi containing omega-3 fatty acids from algal oil was prepared by the addition of oil-in-water emulsions or bulk oil. Emulsion and bulk oil were added separately to surimi to provide approximately 500 mg of omega-3 fatty acids per serving of surimi (85 g). Addition of the emulsion had no effect on surimi gel strength, whereas bulk oil decreased gel strength an average of 31%. All surimi treatments containing algal oil increased in Hunter b values due to the presence of carotenoids in the oil. Among cryoprotectants, sodium tripolyphosphate was the major surimi additive responsible for retarding the formation of lipid hydroperoxides and thiobarbituric acid reactive substances (TBARS). Lipid hydroperoxide and TBARS formation was lower in surimi containing bulk oil compared to surimi with emulsified oil. Both EDTA and lipid soluble antioxidants were able to decrease lipid oxidation in surimi fortified with omega-3 fatty acids. This suggests that surimi containing nutritionally beneficial omega-3 fatty acids could be developed with good oxidative stability and gel strength.     

Effect of low pH on the susceptibility of isolated cod (Gadus morhua) microsomes to lipid oxidation

During the extraction of muscle to produce protein isolates by acid or alkali solubilization, membranes are exposed to abnormally low or high pH. Low but not high pH treatment induces rapid oxidation of membrane phospholipids in the presence of hemoglobin. The goal of this research work was to study the oxidative stability of microsomes under the conditions met during acid solubilization. Isolated microsomes from cod muscle were used as a model system. At pH 5.3 or lower, 99% of isolated cod membranes sedimented at low centrifugation speeds. Isolated membranes that were exposed to pH 3.0 were less susceptible to hemoglobin-mediated lipid oxidation. Cod hemoglobin exposed to pH 3 was rendered less pro-oxidative than the untreated cod hemoglobin. However, when microsomes and hemoglobin were together exposed to low pH, oxidation was promoted. Citric acid and calcium chloride, as well as press juice isolated from cod muscle, were able to inhibit lipid oxidation of microsomal suspensions.     

Flavor and quality characteristics of salted and desalted cod (Gadus morhua) produced by different salting methods

Flavor characterization and quality of salt-cured and desalted cod (Gadus morhua) products was studied using sensory analysis and gas chromatography techniques. The products were produced in Iceland using two different processing methods (filleting and splitting) and three different salting procedures, i.e., the old single-step kench salting or a multistep procedure, and presalting (injection and brine salting or only brine salting), which was followed by kench salting. The main difference observed was between fillets and split fish, where the split fish was darker and had stronger flavor characteristics. Comparison of different salting procedures showed that the use of presalting improved the appearance of the salted products, which can be described as increased lightness and reduced yellowness of the products. In the same products, the intensity of curing flavors was milder, as described by sensory analysis and key aroma compounds. Derivatives from lipid and protein degradation contribute to the characteristic flavor of the salted products.     

Effect of antioxidants, citrate, and cryoprotectants on protein denaturation and texture of frozen cod (Gadus morhua)

To investigate the role of antioxidants and cryoprotectants in minimizing protein denaturation in frozen lean fish, cod fillets were treated with either antioxidants (vitamin C (500 mg kg(-1)) or vitamin C (250 mg kg(-1)) + vitamin E (250 mg kg(-1))), antioxidants (vitamins C + E 250 mg kg(-1)each) with citrate (100 mg kg(-1)), cryoprotectants (4% (w/w) sucrose + 4% (w/w) sorbitol), or a mixture of antioxidants (vitamins C + E 250 mg kg(1)), citrate (100 mg kg(-1)), and cryoprotectants (sucrose 40 g kg(-1) + sorbitol 40 g kg(-1)). Untreated and treated fish samples were stored at -10 degrees C; cod fillets stored at -30 degrees C were used as a control. Stored frozen samples were analyzed at intervals for up to 210 days for changes in protein extractability, thermodynamic parameters (transition temperature T(m) and enthalpy DeltaH), structure by FT-Raman spectroscopy, and rheological properties by large and small deformation tests. Results indicated that protein denaturation and texture changes were minimized in the presence of cryoprotectants, as well as in the presence of antioxidants with citrate, antioxidants alone, or the mixture of antioxidants, citrate, and cryoprotectants. In the presence of increased formaldehyde levels in fish treated with vitamin C, toughening was still lower compared to that of the -10 degrees C control due to the antioxidant property of vitamin C. Thus, ice crystal formation and lipid oxidation products are the major factors that cause protein denaturation in lean frozen fish, and antioxidants in addition to cryoprotectants can be used to minimize toughness.     

pH-induced shift in hemoglobin spectra: a spectrophotometeric comparison of atlantic cod ( Gadus morhua ) and mammalian hemoglobin

Due to a pH-sensitive effect in many fish hemoglobins (Hb), analytical errors may occur when mammalian Hb is used as a standard in quantitative spectrophotometric multicomponent analysis of fish blood. The aim of this work was to examine differences in the optical spectra of mammalian (human) and fish (farmed Atlantic cod) Hb subjected to pH 7.4 and 6.5. The absorption spectra of the common derivatives, deoxy- (HHb), oxy- (OHb), carboxy- (COHb), and methemoglobin (metHb), were determined in the spectral range of 450-700 nm. The metHb spectra of fish differed considerably from the corresponding human Hb spectra, whereas only minor differences in OHb, HHb, and COHb were found. Cod Hb was significantly (P < 0.05) influenced by a drop in pH compared to mammalian Hb. This resulted in deoxygenation of the Hb and increased autoxidation. For human Hb, a pH-independent isosbestic point in the spectra of OHb, HHb, and metHb at 523 nm was found. This isosbestic point was not found in the absorption spectra of cod Hb. In conclusion, spectra of cod metHb and human metHb behave differently. This must thus be taken into account in spectrophotometric multicomponent analysis. Ideally, Hb in muscle or blood should be determined by comparison to a standard made from the same species.     

Effects of oxidized dietary oil and vitamin E supplementation on lipid profile and oxidation of muscle and liver of juvenile atlantic cod (Gadus morhua)

The effects of oxidized dietary lipid and the role of vitamin E on lipid profile, retained tocopherol levels, and lipid oxidation of juvenile Atlantic cod (Gadus morhua) were evaluated following a 9-week feeding trial. Four isonitrogenous experimental diets containing fresh or oxidized (peroxide value of 94 mequiv/kg) fish oil with or without added vitamin E (alpha-tocopherol or mixed tocopherols) were fed to juvenile cod in duplicate tanks. There was no significant (P > 0.05) influence on major lipid classes of cod liver and muscle by diet with the exception of sterols. Sterols content was increased in liver but decreased in muscle by oxidized dietary oil in the absence of vitamin E. Dietary vitamin E supplementation decreased the sterols level in cod liver but with no significant (P > 0.05) effect on their level in the muscle. Fatty acid composition varied between lipid fractions in muscle tissue and was affected by the diet. Oxidized oil significantly (P < 0.05) decreased the deposition of alpha-tocopherol in liver but not in muscle. gamma- and delta-Tocopherols from dietary tocopherol mixtures were retained at very low levels in liver, but higher retention was observed in muscle tissue. The oxidative state of both liver and muscle, as measured by the 2-thiobarbituric acid reactive substances (TBARS) and headspace propanal, negatively correlated with tissue vitamin E levels. It is suggested that oxidized oil affected juvenile Atlantic cod by causing vitamin E deficiency in certain tissues and that these effects could be alleviated by supplementation of a sufficient amount of dietary vitamin E. The results also indicate that mixed tocopherols were good antioxidants for Atlantic cod, although less effective than alpha-tocopherol alone in many tissues with the exception of muscle, where gamma- and delta-tocopherols were deposited at relatively high levels.     

Water distribution in brine salted cod (Gadus morhua) and salmon (Salmo salar): a low-field 1H NMR study

Low-field (LF) (1)H NMR T 2 relaxation measurements were used to study changes in water distribution in lean (Atlantic cod) and fatty (Atlantic salmon) fish during salting in 15% NaCl and 25% NaCl brines. The NMR data were treated by PCA, continuous distribution analysis, and biexponential fitting and compared with physicochemical data. Two main water pools were observed in unsalted fish, T 21, with relaxation times in the range 20-100 ms, and T 22, with relaxation times in the range 100-300 ms. Pronounced changes in T 2 relaxation data were observed during salting, revealing changes in the water properties. Salting in 15% brine lead to a shift toward longer relaxation times, reflecting increased water mobility, whereas, salting in saturated brines had the opposite effect. Water mobility changes were observed earlier in the salting process for cod compared to salmon. Good linear correlations ( F 相似文献   

Lipid fractions with aggregatory and antiaggregatory activity toward platelets in fresh and fried cod (Gadus morhua): correlation with platelet-activating factor and atherogenesis

Cod (Gadus morhua) is a popular part of the diet in many countries on both sides of the North Atlantic; in most cases it is consumed fried. In this study, total lipids of cod muscle were separated into neutral and polar lipids, which were further fractionated by HPLC. The lipid fractions were tested in vitro, against washed rabbit platelets, for the probable existence of lipid compounds that either exhibit an action similar to that of platelet-activating factor (PAF) or inhibit the action of PAF. The platelet bioassay was used to evaluate total lipids, total polar lipids, and total neutral lipids, before any further separation. Detection of these compounds in fresh and fried cod could be used to evaluate the nutritional value of this important fish. The in vitro biological study of lipids showed that in fresh cod lipid fractions, ranges of PAF-like and anti-PAF-like activities were present, whereas in fried cod lipid fractions, both neutral and polar, anti-PAF activities were mainly observed. Because it has already been reported that PAF is involved in atheromatosis generation, the existence of PAF inhibitors in cod may contribute to the possible protective role of fish, in this case cod, against atherosclerosis.     

Effect of chitosan and chitin on the separation of membranes from proteins solubilized by pH shifts using cod (Gadus morhua)

Studies with isolated membranes and isolated membranes suspended in muscle proteins solubilized at pH 3 showed that mixing chitosan and membranes at this low pH followed by a pH adjustment to 10.5 could sediment membranes effectively at 4000 g. In the solubilized muscle homogenate, the effectiveness of membrane removal by chitosan at 4000 g for 15 min was molecular weight dependent. About 80% of the phospholipids and 28% of proteins were sedimented from solubilized muscle homogenate by mixing muscle homogenate (10 g of muscle tissue homogenized with 90 mL of distilled water) with 10 mL of MW 310-375 k chitosan (10 mg/mL in 0.1 N HCl) before solubilizing it at pH 10.5, whereas 55% of the phospholipids and 12% of proteins were sedimented by mixing muscle homogenate with the MW 310-375 k chitosan before solubilizing the homogenate at pH 3. Low molecular weight chitosans (at MW 1k or 33k) showed little effect on membrane sedimentation under the same conditions. Chitin was not useful for removing membranes at either pH 3 or 10.5, whether added before or after pH adjustment.     

Hemoglobin-mediated lipid oxidation and compositional characteristics of washed fish mince model systems made from cod (Gadus morhua), herring (Clupea harengus), and salmon (Salmo salar) muscle

The use of washed cod light muscle minces in mechanistic studies of hemoglobin (Hb)-mediated fish lipid oxidation has largely increased in the past 5 years. Although cod light muscle has a low level of intrinsic lipid oxidation catalysts, a prerequisite for a good oxidation model system, we believe it cannot fully mimic the oxidation kinetics taking place in other fish species being more susceptible to lipid oxidation. The aim of this study was to systematically investigate whether washed mince model systems useful in Hb-mediated oxidation studies could be prepared also from herring (Clupea harengus) and salmon (Salmo salar) light muscles. The kinetics of oxidation in the washed models was measured during ice storage (+/-Hb), and the results were related to compositional differences. Minces from cod, herring, and salmon light muscles were washed 3 times with 3 volumes of water and buffer. A 20 microM portion of Hb and 200 ppm streptomycin was then added, followed by adjustment of pH and moisture to 6.3 and 86%, respectively. Samples with or without Hb were then stored on ice, and oxidation was followed as peroxide value (PV), rancid odor, redness (a*) loss and yellowness (b*). Prior to storage, all minces and models were also analyzed for total lipids, fatty acids, alpha-tocopherol, proteins, Hb, Fe, Cu, and Zn. Hb-mediated lipid oxidation appeared within 2 days on ice in all models. Small differences in the oxidation rates ranked the models as herring > cod > salmon. These differences were ascribed to more preformed peroxides and trace elements in the herring model, and more antioxidants in the salmon model. Controls, without Hb, stayed stable in all cases except herring, where a very slight oxidation appeared, especially if the herring raw material had been prefrozen. In conclusion, fattier fish like dark muscle species and salmonoids are useful for making washed mince model systems and would be a better choice than cod if there is an interest in the oxidation kinetics of such species.     

Production of cod (Gadus morhua) muscle hydrolysates. Influence of combinations of commercial enzyme preparations on hydrolysate peptide size range

The complement of enzyme activities of a selection of commercial protease preparations were determined using fluorogenic substrates. Alcalase was used in combination with other commercial enzyme preparations to produce cod muscle (Gadus morhua) hydrolysates. Each muscle hydrolysate was characterized with respect to the percentage degree of hydrolysis (DH %), peptide molecular weight range, and free amino acid content. The enzyme preparations containing predominantly protease or endopeptidase activities achieved high DH % and produced significant amounts of peptides below a molecular weight of 3000. Alcalase combined with exopeptidase-rich preparations produced hydrolysates rich in low-molecular-weight peptides. Selecting combinations of enzyme preparations with complementary activity profiles could be used to manipulate the peptide molecular weight profile of hydrolysates.     

Characterization of volatile compounds in chilled cod (Gadus morhua) fillets by gas chromatography and detection of quality indicators by an electronic nose

Volatile compounds in cod fillets packed in Styrofoam boxes were analyzed during chilled storage (0.5 degrees C) by gas chromatography (GC)-mass spectrometry and GC-olfactometry to screen potential quality indicators present in concentrations high enough for detection by an electronic nose. Photobacterium phosphoreum dominated the spoilage bacteria on day 12 when the fillets were rejected by sensory analysis. Ketones, mainly 3-hydroxy-2-butanone, were detected in the highest level (33%) at sensory rejection, followed by amines (TMA) (29%), alcohols (15%), acids (4%), aldehydes (3%), and a low level of esters (<1%). The electronic nose's CO sensor showed an increasing response with storage time coinciding with the production of ethanol and 2-methyl-1-propanol that were produced early in the storage, followed by the production of 3-methyl-1-butanol, 3-methyl-butanal, 2,3-butandiol, and ethyl acetate. Lipid-derived aldehydes, like hexanal and decanal, were detected in similar levels throughout the storage time and contributed to the overall sweet odors of cod fillets in combination with other carbonyls (3-hydroxy-2-butanone, acetaldehyde, 2-butanone, 3-pentanone, and 6-methyl-5-heptene-2-one).     

Dietary lipid source modulates in vivo fatty acid metabolism in the freshwater fish, Murray cod (Maccullochella peelii peelii)

The aim of the present investigation was to quantify the fate of C18 and long chain polyunsaturated dietary fatty acids in the freshwater fish, Murray cod, using the in vivo, whole-body fatty acid balance method. Juvenile Murray cod were fed one of five iso-nitrogenous, iso-energetic, semipurified experimental diets in which the dietary fish oil (FO) was replaced (0, 25, 50, 75, and 100%) with a blended vegetable oil (VO), specifically formulated to match the major fatty acid classes [saturated fatty acids, monounsaturated fatty acids, n-3 polyunsaturated fatty acids (PUFA), and n-6 PUFA] of cod liver oil (FO). However, the PUFA fraction of the VO was dominated by C18 fatty acids, while C20/22 fatty acids were prevalent in the FO PUFA fraction. Generally, there was a clear reflection of the dietary fatty acid composition across each of the five treatments in the carcass, fillet, and liver. Lipid metabolism was affected by the modification of the dietary lipid source. The desaturation and elongation of C18 PUFAs increased with vegetable oil substitution, supported by the occurrence of longer and higher desaturated homologous fatty acids. However, increased elongase and desaturase activity is unlikely to fulfill the gap observed in fatty acid composition resulting from decreased highly unsaturated fatty acids intake.     

Bioactive compounds in cod (Gadus morhua) products and suitability of 1H NMR metabolite profiling for classification of the products using multivariate data analyses

This work investigates the suitability of (1)H NMR spectroscopy to identify the fate of some bioactive compounds in seafood submitted to several processing conditions and examines the possibility of using (1)H NMR spectroscopy profiling to classify such products. Perchloric acid extracts of cod white muscle from newly killed and (i) unprocessed, (ii) boiled, and (iii) fried fillets and from (iv) frozen fillets, (v) the frozen fillets after thawing, and (vi) their drip loss and from (vii) rehydrated cod klippfish (n = 5) were analyzed by 500 MHz (1)H NMR spectroscopy. It was possible to identify taurine, betaine, anserine, creatine, and trimethylamine oxide (TMAO) in all extracts examined, and frozen fish was recognizable by the presence of dimethylamine (DMA). None of the heating procedures seemed to induce the loss of bioactive compounds from the fillet, but freezing and thawing did: the compounds were lost in what is known as drip loss. About 80% of the samples were correctly classified using a probabilistic neural network procedure having as inputs the scores of the first 20 principal components of the principal component analysis of a selected region of the NMR spectra.     

Effects of temperature and life stage on the fatty acid composition of Collembola

Fatty acid (FA) analysis is used as a promising tool to investigate trophic interactions in soil food webs. The FA profile of neutral lipids in consumers is affected by the diet, and the occurrence and amount of certain FAs can reflect feeding strategies. We investigated the lipid composition of the Collembola Folsomia candida, Heteromurus nitidus and Protaphorura fimata with the fungus Chaetomium globosum as food source. The impact of environmental temperature and life stage was assessed, with special respect to linoleic acid (18:2ω6,9) as a marker FA for fungal feeding. In all Collembola species the ratio of C16/C18 in neutral lipid fatty acids (NLFAs) increased with decreasing temperature. In the NLFAs of F. candida and H. nitidus the Unsaturation Index and the amount of 18:2ω6,9 decreased with temperature, whereas in P. fimata effects were the opposite. The composition of phospholipid fatty acids (PLFAs) differed between species, but was little affected by temperature. The degree of unsaturation in NLFAs increased with the age of Collembola, mainly due to higher amounts of 18:2ω6,9 and a lower proportion of 18:1ω9. The biomarker linoleic acid represented over 20% of FAs in all fungal feeding Collembola. Despite considerable influence of temperature and life stage on its proportion, the amount was always higher than in individuals reared on other diets. This suggests that linoleic acid can serve as marker for fungal feeding independent of such physiological variations in Collembola.     

Effects of nitric oxide on fatty acid composition in peach fruits during storage

Peach (Prunus persica (L.) Batsch., cv. Feicheng) fruits were fumigated with 0, 5, 10, and 15 microL/L nitric oxide (NO), and the effects of NO on rot index, ion leakage, and the lipid compositions in peach fruits were studied. The results showed that the treatment with 10 microL/L NO significantly increased the contents of the palmitoleic (16:1), oleic (18:1), and linolenic (18:3) acid in phospholipid (PL), while it decreased the contents of linoleic (18:2) acid in PL. The content of monoacylglycerol (MG) in fruit fumigated with 10 microL/L NO were higher than in the control but lower in fruit fumigated with 15 microL/L NO. The treatment with 10 microL/L NO increased the content of MG, decreased the contents of diacylglycerol (DG) and triacylglycerol (TG). However, there was no significant difference in the contents of free fatty acid (FFA). The compositions of MG, DG, TG, and FFA in peach fruit were also changed by the treatment with 10 microL/L NO. It could preserve the content of 18:3, maintain the integrity of membrane, and prevent the softening and rotting of peach fruit. The content of 18:1 was detected in PL and FFA but not in MG, DG, and TG. This might be due to the different compositions of MG, DG, and TG.     

Validation of a tRNA-Glu-cytochrome b key for the molecular identification of 12 hake species (Merluccius spp.) and Atlantic Cod (Gadus morhua) using PCR-RFLPs, FINS, and BLAST

The goal of this study was to develop a diagnostic key for hake meat to solve the limitations of previous identification methodologies, mainly related to the high degradation of the DNA recovered from processed foods. We describe the development of two molecular tools based on polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphisms of the cytochrome b gene, respectively, to identify DNA from 12 hake species in commercial products. The first assay is an exclusion test consisting of the PCR amplification of a 122 bp fragment using nested primers interspecifically conserved in Merluccius spp. and in Gadus morhua. This 122 bp amplicon, being the shortest one so far designed for hake DNA, is a useful traceability tool for highly degraded samples because its sequence contains enough interspecific diagnostic variation to identify 10 hake species and cod and has been successfully amplified from most commercial products so far tested. The second identification key follows a positive outcome of the exclusion test and consists of the PCR amplification of a 464-465 bp fragment and its digestion with three restriction enzymes whose targets map at interspecifically nonconserved sites of the cytochrome b. The key presented here has passed through a rigorous methodological calibration including its testing for genus specificity, its validation on a large number of authenticated sample types from each species range, and its implementation with a maximum likelihood method for the assignment of unknown samples. Together, these two procedures constitute the most complete molecular key so far developed for Merluccius spp., which is optimal for routine identification of hakes in large commercial samples at a reasonable cost-time ratio.     

Ultrastructure of the myofibrillar component in cod (Gadus morhua L.) and hake (Merluccius merluccius L.) stored at -20 degrees C as a function of time.

Transmission electron microscopy and image analysis techniques were used to study the ultrastructure of the myofibrillar component in cod and hake muscle stored at -20 degrees C for varying periods of time. Cod muscle showed a deformation of the hexagonal array of thick filaments with the storage time, reflected in an increase in the eccentricity value, a parameter defined to measure changes in the ratio of maximum to minimum hexagon diameter, and an increase in the cross-linkings between the filaments. Degradation of cod thick filaments leading to detachment was also visible upon prolonged storage. In hake muscle significant changes were not found in the arrangement and morphology of thick filaments during frozen storage, suggesting a high incidence of intrafilament aggregation. The ultrastructural differences in the array of thick filaments between species were accompanied by a difference in the textural measurements.